Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for the study assembly and maintenance of human kinetochore as well as for gene therapy, screening of anticancer drugs and biotechnology. We previously constructed a synthetic HAC (tetO-HAC) allowing tethering of its kinetochore by different chromatin modifies fused with the tet-repressor protein. The tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, such as the tTS, to its centromeric tetO sequences. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the tetO-HAC. In separate experiments, a platform with multi-integrase recombination sites has been inserted into tetO-HAC and has been successfully used for a gene assembly in the HAC. A new tetO-HAC system has several notable advantages that set it apart from other artificial chromosome-based systems, including assembly of unlimited number of genomic DNA segments and the opportunity to remove mis-incorporated DNA segments. Work is in progress to assemble synthetic nucleolar organizer region (NOR) in the HAC using TAR-isolated human rDNA units. Such a HAC module will be used to investigate the requirements for nucleolar location of rDNA repeats and effect of copy number of rDNA units on cell proliferation and stress response. Note that despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. In our recent work, we recovered ribosomal DNA segments from a single human chromosome using transformation-associated recombination (TAR) cloning in yeast. Accurate long-read sequencing of 13 isolates covering 0.82 Mb of the chromosome 21 rDNA complement revealed substantial variation among tandem repeat rDNA copies and several palindromic structures. These clones revealed 101 variant positions in the 45S transcription unit and 235 in the intergenic spacer sequence. Most of the 45S variants were confirmed in independent whole-genome or RNA-Seq data. The large number of variants observed provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human diseases. The loading of different variants of a human rDNA unit into the tetO-HAC may help to clarify the peculiarity of PolI transcription machinery as well as the mechanism by which rDNA units are selected and targeted for chromatin changes leading to heterochromatinization in NORs. The assembly of the entire rDNA locus in the HAC vector has multiple applications in functional genomics and biomedicine. In our previous work, we demonstrated that the tetO-HAC is a highly versatile model system to study centrochromatin and its impact on kinetochore structure and function in human cells. However, this HAC comprises only one type of alphoid repeats and therefore cannot be used to study a role of heterochromatin in kinetochore function because both centrochromatin and heterochromatin domains in the HAC are assembled on the same sequences, thus precluding specific tethering of chromatin effectors only to one domain. To investigate a role of heterochromatin domains in kinetochore function, we have constructed a new HAC containing two different synthetic alphoid arrays, a tetO-containing array with CENP-B boxes and a new synthetic alphoid array containing recognition sites for the lacO operator and yeast transcriptional factor Gal4. Notably, this array contains no CENP-B boxes. As known, lack of CENP-B boxes precludes CENP-A assembly on transfecting alphoid DNA arrays. The novel hybrid HAC has more regular structure compared to the original synthetic tetO-HAC previously constructed from the dimer containing incorporated tetO sequence. More importantly, ChIP analysis demonstrated CENP-A assembly on tetO alphoid arrays in the HAC. Thus, in the HAC, a heterochromatin domain is formed on the lacO/gal4 alphoid arrays. This configuration could be used to look at whether the heterochromatin or centrochromatin spreads into another array, and whether they can be forced to spread laterally by targeting chromatin modifiers. We have also applied our tetO-HAC for measuring chromosome instability (CIN) in human cells. Whole-chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a characteristic feature of most types of cancer, thus distinguishing them from their normal counterparts. Although CIN is generally considered a driver of tumor growth, a threshold level exists whereby further increase in CIN frequency becomes a barrier against tumor growth and therefore can be exploited therapeutically. However, drugs known to increase CIN beyond this therapeutic threshold are currently few in number. In our previous work, we have developed a new quantitative assay for measuring CIN based on the use of a non-essential HAC carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows measurement of HAC loss rate in response to drug treatment by routine flow cytometry. We used this assay to rank more than 150 anticancer drugs on their effect on HAC loss. The strongest effect was observed for microtubule-stabilizing agents and inhibitors of topoisomerase TOP1, developed in our branch. We also demonstrated the utility of the assay to detect increase of CIN after siRNA depletion of known genes controlling chromosome transmission. In our recent work, we modified EGFP-HAC and converted the original assay into high-throughput CIN screen of chemical libraries and siRNA libraries of human genes. Analysis of siRNAs targeting each of 720 human protein kinase genes revealed 27 CIN genes with no information on their role in chromosome transmission. Each of these new CIN genes may be considered as a new target for cancer therapy. The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In our recent work, we designed a new protocol to screen for, and rank the efficacy of, compounds specifically targeting telomeres and telomerase. The protocol is based on the use of two isogenic cell lines containing a circular HAC (lacking telomeres) and a linear HAC (containing telomeres) marked with the EGFP transgene: compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. We applied this novel dual-HAC assay to rank a set of known and newly developed compounds, including G4 ligands. Among the latter group, we found two compounds -Cu tt Py and Pt tt Py- that induce a high rate of linear HAC loss with no significant effect on the mitotic stability of a circular HAC. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges in late mitosis and cytokinesis. Further cytological analysis showed that chromosome loss after Pt tt Py or Cu tt Py treatment correlated with the induction of telomere-associated DNA damage. Identification and ranking of compounds that greatly increase chromosome mis-segregation rates as a result of telomere dysfunction may expedite the development of new therapeutic strategies for cancer treatment.